Expression of the human plasma membrane Ca2+-ATPase isoform 4xb in Saccharomyces cerevisiae. Acidic lipids are necessary for the reactivation of the purified enzyme

CURA CAROLINA; CORRADI GERARDO RAUL; RINALDI DEBORA ELENA; ADAMO HUGO PEDRO

Ahrus, Dinamarca

Conferencia; 12th International ATPase Conference Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease; 2008

Universidad de Ahrus

The cDNA coding for the human plasma membrane Ca2+ pump (isoform 4xb, hPMCA4xb) was cloned into the pMP625 vector and expressed in S. cerevisiae under the control of the strong PMA1 promoter. Yeast membranes were solubilized with detergent C12E10 and 20% glycerol without the addition of exogenous lipids, and the PMCA protein was purified by calmodulin-affinity chromatography. The mobility of the recombinant hPMCA4xb purified from yeasts (yPMCA) in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of PMCA purified from human erythrocytes (ePMCA). The function of the recombinant enzyme was investigated by measuring the Ca2+-dependent ATPase activity. In mixed micelles of phosphatidylcholine-detergent the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA. By contrast, under the same conditions the ePMCA had a Ca2+-ATPase activity of 3.8 μmol/mg/min. At saturating concentrations of Ca2+, the addition of acidic lipids increased about 3 fold the activity of the ePMCA while it caused about 12 fold increase in the activity of the yPMCA. It is well known that acidic lipids activate the ePMCA interacting with the C-terminal autoinhibitory domain and mimicking the activation produced by calmodulin. Alternatively it has been shown that the interaction of acidic lipids with a region in the "A domain" can activate the ePMCA by shifting the apparent affinity for Ca+2 to a value even lower that that attained in the presence of calmodulin. However, judged by the increase in the level of phosphoenzyme, a novel effect of acidic lipids increasing the total amount of active enzyme was involved in the reactivation of the detergent solubilized yPMCA. These results show that the function of yPMCA is highly sensitive to delipidation and reveal that the restitution of acidic lipids is needed for a fully functional enzyme. This work was supported in part by the University of Buenos Aires (UBA, Grant B029), by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Grant PIP 6168), and by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Grant BID 1728 OC-AR PICT 15-25965). Presenting author: